This is an application for supplemental funds to cover the third, and final, year of a research project begun 1.5 years ago. As stated in the parent application, "the overall objective of this proposal is to examine the molecular mechanisms by which divalent cations such as calcium interact with both artificial bilayers and the bilayer component of biological membranes". More specifically, we are investigating whether the Stern equation can describe the adsorption of divalent cations to membranes. We are making independent measurements of both the number of adsorbed cations and the electrostatic surface potential. The number of adsorbed calcium ions are measured by ion selective electrodes and the number of adsorbed cobalt ions by P31 NMR. The electrostatic potential is determined by zeta potential measurements on multilamellar vesicles, fluorescent probe measurements with TNS on sonicated vesicles and conductance measurements with carriers on planar (Montal-Mueller) bilayer membranes. When the project began little money was requested for the third year because we did not know whether our techniques would yield data that could be interpreted easily. The experimental data presented in the body of the application and in the attached publications demonstrate that all our techniques are functioning well. In agreement with the previous observations of Abramson, Puskin, Rand, Nir, Kurland and coworkers, we have obtained evidence that monovalent cations adsorb to negative lipids. In contrast to the assumptions, but not the data, of other workers, our measurements reveal that divalent cations form 1:1 complexes with negative lipids. All our data are consistent with an extremely simple model, the Gouy-Chapman-Stern formulation, and funds are requested for supplies and technical help to complete the project.